Team:HokkaidoU Japan/Notebook/September23

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*Bac Vector の精製
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*Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification
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*AraC+RBS+pSB1A3のコロニーPCR
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*Colony PCR of AraC+RBS+pSB1A3
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*DH5αとMG1655へのBac Vecterのelectroporetion
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*Electroporetion of BAC plasmid into DH5α MG1655
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== Bac Vecter Purification ==
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= Bac Vecter Purification =
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miniprepはQiagenのminiprep kit,qiaprepを用いて行った。
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Used Qiagen miniprep kit, qiaprep for miniprep
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1)昨日から培養してあったBac Vector保有株を1.3ml,1.5mlエッペンチューブに移した。
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#Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube
 +
#Centrifuged at 4C, 15000 rpm for 1 min
 +
#Discarded the supernatant and added remaining solution.
 +
#Centrifuged at 4C, 15000 rpm for 1 min
 +
#Discarded the supernatant
 +
#Suspended on 250 uL of Buffer P1
 +
#Added 250 ul Buffer P2 inverted few times to mix, solution turned green
 +
#Added 350 ul Buffer N3 mixed by inversion, precipitation apeared
 +
#Centrifuged at 4C, 13000 rpm for 10 min
 +
#Transfered the supernatant to filtration column
 +
#Centrifuged at 4C, 13000 rpm for 1 min
 +
#Discarded the flow-through
 +
#added 500 uL of Buffer PB  to filtration column
 +
#Centrifuged at 4C, 13000 rpm for 1 min
 +
#Discarded the flow-through centrifuged for 1min to remove remaining buffer
 +
#Transfered filtration column to a new 1.5 ml tube
 +
#Resuspended on 50 ul of TE and incubated at RT for 1min
 +
#Centrifuged at 4C, 13000 rpm for 1 min
 +
#Stored at -20C
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2)4C、15000rpmで1min遠心を行った。細胞塊が沈殿した。
 
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3)上澄みを捨て、残りのサンプルをチューブに移した。
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= Colony PCR of AraC+RBS+pSB1A3 =
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*Selected 16 colonies at random, and PCRed them.  
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4)4C、15000rpmで1min遠心を行った。細胞塊が沈殿した。
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*PCR mix used is shown in the table bellow
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+
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5)上澄みを捨て、4℃で保存していたBuffer P1を250 ul加え、voltexにかけた。細胞塊は見えなくなった。
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-
 
+
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6)Buffer P2を250 ul加え、静かに数回転倒混和した。溶液の色が緑色に変化した。
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+
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7)Buffer N3を350 ul加え、すぐに数回転倒混和した。溶液が白濁した。
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+
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8)4C,13000rpmで10min遠心した。
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+
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9)上澄みをピペットマンで専用のカラムに移し、4C,13000rpm,1min遠心した。
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+
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10)下澄み液を捨て、500 ulのBuffer PBを加えて4C,13000rpm,1minで遠心を行った。
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+
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11)下澄み液を捨て、さらに1min遠心した。
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+
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12)新しい1.5 ml チューブにカラムを移し、TEを50 ul加えて1min放置した。
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+
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13)4C,13000rpm,1min遠心を行った。
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14)-20Cで凍結保存した。
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== AraC+RBS+pSB1A3のコロニーPCR ==
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#コロニーを無作為に16個選び、番号を振った。
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#0.5 mLチューブを16本用意し、それぞれにDWを10 uLずつ分注した。
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#チューブに番号を振り、対応するコロニーを懸濁した。
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#コロニーPCR溶液を以下の組成に従って調整した。
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{|style="text-align:center; margin-left:100px;" class="protocol"
{|style="text-align:center; margin-left:100px;" class="protocol"
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|-
|-
|Taq Master Mix
|Taq Master Mix
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|90 uL
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|80 uL
|-
|-
|Ex-F
|Ex-F
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|1.8 uL
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|1.6 uL
|-
|-
|Ps-R
|Ps-R
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|1.8 uL
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|1.6 uL
|-
|-
|style="border-top:1px solid #996;"|'''Total'''
|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''93.6 uL'''
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|style="border-top:1px solid #996;"|'''83.2 uL'''
|}
|}
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 +
[[Image:9.23 coloP.JPG‎|200px|right|thumb|Result of colony PCR]]
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 +
*electrophoresed the samples
 +
 +
1 lane -> Marker λ/HindIII EcoRI 5 uL
 +
 +
2~8 lane -> colony No.1~7
 +
 +
9 lane -> Marker λ/HindIII EcoRI 5 uL
 +
 +
10~16 lane -> colony No.8~14
 +
 +
 +
Colony No.15,16 didn't electrophorese.
 +
 +
 +
*Colony No.1 will have the plasmid,so we put the sample into 2 mL LB added 2 uL Ampicillin(100 ug/uL).Incubated it in a shaker at 37C, 180 rpm.

Latest revision as of 18:02, 27 October 2010

  • Amplifiable BAC plasmid (BBa_J61031) purification
  • Colony PCR of AraC+RBS+pSB1A3
  • Electroporetion of BAC plasmid into DH5α MG1655

Bac Vecter Purification

Used Qiagen miniprep kit, qiaprep for miniprep

  1. Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube
  2. Centrifuged at 4C, 15000 rpm for 1 min
  3. Discarded the supernatant and added remaining solution.
  4. Centrifuged at 4C, 15000 rpm for 1 min
  5. Discarded the supernatant
  6. Suspended on 250 uL of Buffer P1
  7. Added 250 ul Buffer P2 inverted few times to mix, solution turned green
  8. Added 350 ul Buffer N3 mixed by inversion, precipitation apeared
  9. Centrifuged at 4C, 13000 rpm for 10 min
  10. Transfered the supernatant to filtration column
  11. Centrifuged at 4C, 13000 rpm for 1 min
  12. Discarded the flow-through
  13. added 500 uL of Buffer PB to filtration column
  14. Centrifuged at 4C, 13000 rpm for 1 min
  15. Discarded the flow-through centrifuged for 1min to remove remaining buffer
  16. Transfered filtration column to a new 1.5 ml tube
  17. Resuspended on 50 ul of TE and incubated at RT for 1min
  18. Centrifuged at 4C, 13000 rpm for 1 min
  19. Stored at -20C


Colony PCR of AraC+RBS+pSB1A3

  • Selected 16 colonies at random, and PCRed them.
  • PCR mix used is shown in the table bellow
Reagent Amount
Taq Master Mix 80 uL
Ex-F 1.6 uL
Ps-R 1.6 uL
Total 83.2 uL
Result of colony PCR
  • electrophoresed the samples

1 lane -> Marker λ/HindIII EcoRI 5 uL

2~8 lane -> colony No.1~7

9 lane -> Marker λ/HindIII EcoRI 5 uL

10~16 lane -> colony No.8~14


Colony No.15,16 didn't electrophorese.


  • Colony No.1 will have the plasmid,so we put the sample into 2 mL LB added 2 uL Ampicillin(100 ug/uL).Incubated it in a shaker at 37C, 180 rpm.